Salt and pepper staining patterns for LAT zap70 in allergic bullous drug reaction

Original Articles / Prace Oryginalne
Abreu Velez Ana Maria1, Jackson Billie L.2, Howard Michael S.1
Georgia Dermatopathology Associates, Atlanta, Georgia, USA.
[email protected]
Billie L. Jackson, M.D., LLC, Macon, Georgia, USA.
N Dermatol Online. 2011; 2(3): 104-107
Date of submission: 10.02.2011 / acceptance: 19.03.2011
Conflicts of interest: None
Background. The term bullous drug eruption refers to clinically adverse drug reactions that result in fluid-filled blisters or bullae.
Blistering can be elicited by multiple medications, prescribed or over-the-counter, natural or synthetic. Case Report: A 78-year-old
female was evaluated for the presence of a rapidly appearing, diffuse rash with vesicles, bullae and abdominal edema. Methods: Skin
biopsies for hematoxylin and eosin examination, as well as for direct immunofluorescence and immunohistochemistry analysis were
performed. Results: H&E staining demonstrated a subepidermal blistering disorder. Within the dermis, a mild, superficial, perivascular
infiltrate of lymphocytes, histiocytes and eosinophils was seen. No frank leukocytoclastic vasculitis was appreciated. Direct
immunofluorescence revealed a strong presence of Complement/C3, IgM and fibrinogen in the upper dermal blood vessels. Staining with
LAT, MUM-1, and ZAP-70 was identified in the inflamed vessels, in a delicate salt and pepper pattern. Conclusions: In bullous drug
eruptions, inflammation of the dermal blood vessels without frank leuckocytoclasis is often noted; vascular alterations subjacent to the
blisters are frequently described as nonspecific. We document specific activation markers of the T cell immune response; further
secondary cell signaling pathway molecules are overexpressed in dermal blood vessels, indicative of a complex immune response in
these patients
Wstęp: Termin pęcherzowa wysypka polekowa odnosi się klinicznie niepoŜądanych reakcji polekowych, w których występują
wypełnione płynem większe (bullae) lub mniejsze pęcherze (blister). Pęcherze mogą być wywołane przez wiele leków, przepisanych na
receptę albo bez recepty over-the-counter (OTC), naturalnych lub syntetycznych. Opis przypadku: 78-letnia kobieta została zbadana na
obecność szybko pojawiającej się wysypki z rozproszonymi pęcherzami róŜnej wielkości i obrzęku brzucha. Metody: Wykonano biopsję
skóry z barwieniem hematoksyliną i eozyną, jak równieŜ immunofluorescencję bezpośrednią i analizę immunohistochemiczną. Wyniki:
Barwienie H & E wykazało zaburzenia- subepidermalne pęcherze. W skórze właściwej obserwowano, o łagodnym przebiegu,
powierzchowną, okołonaczyniową infiltrację limfocytów, eozynofili i histiocytów. Nie prawdziwe leukocytoklastyczne zapalenie naczyń
było mile widziane. Immunofluorescencja ujawniła silną obecność Complement/C3, IgM i fibrynogenu w górnych warstwach skórnych
naczyń krwionośnych. Barwienie z LAT, MUM-1 i ZAP-70 było zidentyfikowane w zapaleniu naczyń, jako delikatny wzór soli i
pieprzu. Wnioski: W pęcherzowych wysypkach polekowych, zapalenie naczyń krwionośnych skóry bez prawdziwej leuckocytoclazji
było często zauwaŜalne; zmiany naczyniowe na spodzie pęcherza są często opisywane jako niespecyficzne. Udokumentowaliśmy
swoiste markery aktywacji limfocytów T odpowiedzi immunologicznej; dalsze wtórne ogniwo cząsteczek sygnałowych szlaku są w
nadmiernej ekspresji w naczyniach krwionośnych skóry, co wskazuje na złoŜoną odpowiedź odpornościową u tej pacjentki.
Key words: vasculitides, bullous allergic drug reaction, LAT, ZAP-70, MUM 1, skin.
Słowa klucze: vasculitides, reakcje alergiczne polekowe, LAT, ZAP-70, MUM 1, skóra
Bullous drug reactions (BDRs) may occur
secondary to various medications, both prescribed and
over-the-counter, and natural or synthetic. Blistering may
104 © N Dermatol Online 3.2011
be clinically localized and mild, or widespread and
severe. Blisters may be the major feature of the reaction;
alternatively, blisters may be seen focally, or in localized
areas of a more extensive rash. The reaction may show
features of more than one condition (overlap) or be
clinically unclassifiable. BDRs represent one of the most
dermatopathology, being more prevalent than the classic
nosologic autoimmune cutaneous blistering diseases.
Hematoxylin and eosin staining as well as direct
immunofluorescence (DIF) of the skin, often
demonstrate findings that can be shared by several
diseases, thus being of limited help in establishing a
definitive diagnosis. The typical histologic differential
diagnosis includes 1) a bullous allergic drug reaction, 2)
bullous pemphigoid, or 3) a bullous arthropod bite
reaction. Indirect immunofluorescence (IIF)/salt split
skin studies may be helpful in further distinguishing
between these diagnostic possibilities, if clinically
indicated. We obtained skin biopsies for hematoxylin
immunofluorescence (IIF) with salt split skin studies and
for immunohistochemistry (IHC).
Case report
A 78-year-old female was evaluated for 2
day duration of blisters on the abdomen that were mild to
moderately painful. The patient was taking docusate,
diltiazem, KCl, furosemide, omeprazole, ranitidine,
pravachol and acetaminophen. On physical examination,
the abdomen displayed one intact bulla that was tense,
and showed mild erythema at the base. A second,
adjacent site on the abdomen was consistent with a
ruptured bulla with a denuded surface. There was no
significant crusting to suggest pemphigus vulgaris. The
face, chest and neck showed no involvement. The
patient had an allergy to Codeine. A lesional skin biopsy
was taken for hematoxylin and eosin (H&E) analysis. In
addition, direct and indirect immunofluorescence (DIF,
IIF) and immunohistochemistry (IHC) studies were
DIF and IIF/salt split skin were performed as
previously described. Skin cryosections were prepared,
and incubated with multiple fluorochromes, as
previously reported [1-4].
IHC: Performed as previously described, including
antibodies against the linker of activated T cells (LAT
protein), Zeta-chain-associated protein kinase 70 (ZAP70) and MUM-1(multiple myeloma oncogene-1) protein
demonstrated a subepidermal blistering disorder. Partial
blister re-epithelialization was noted. Within the blister
lumen, eosinophils were present, with occasional
neutrophils and lymphocytes. Mast cells were rare.
Within the dermis, a mild, superficial, perivascular
infiltrate of lymphocytes, histiocytes and eosinophils was
seen. Direct immunofluorescence (DIF) studies were
performed; results were classified as 4+ (very strong
positvity) to (-) negative. Case results were as follows:
IgG(+, focally positive BMZ); IgG3(-); IgG4(-); IgA(-);
IgM (++, at superficial upper dermal vessels); IgD(-);
IgE(-); Complement/C1q(-); Complement/C3 (++),
positive around the upper dermal vessels; albumin(-) and
fibrinogen (++), positive around the upper dermal blood
vessels and inside the subepidermal blister. Antibodies to
human plasminogen were negative (Fig. 1).
Bullous drug eruptions are often diagnosed
clinically, i.e., by careful history and physical
examination. However, in many cases, these reactions
can mimic other diseases [2,4]. The H&E skin biopsy
may help to make the correct diagnosis, but does not
usually help in establishing whether the reaction is druginduced. The presence of a vasculitis-like reaction is
often noted, not fulfilling the diagnostic criteria of a
leukocytoclastic vasculitis.[4-7]. Although we were not
able to appreciate a true leukocytoclastic vasculitis, the
case DIF clearly indicated an immune response against
dermal blood vessels when utilizing FITC conjugated
anti-human fibrinogen and complement/C3. Further, by
IHC we were able to appreciate staining with antibodies
against ZAP-70, LAT and MUM-1 proteins. The protein
encoded by the LAT gene is phosphorylated by ZAP70/SYK protein tyrosine kinases, following activation of
the T-cell antigen receptor (TCR) signal transduction
pathway [8]. The LAT transmembrane protein localizes
to lipid rafts (also known as glycosphingolipid-enriched
microdomains or GEMs), and acts as a docking site for
phosphorylation, this protein recruits multiple adaptor
proteins and downstream signaling molecules into
multimolecular signaling complexes located near the site
of TCR engagement [8]. ZAP-70 is normally expressed
in T cells and natural killer cells and has a critical role in
the initiation of T-cell signaling [9]. ZAP-70 is a member
in the protein-tyrosine kinase family. T lymphocytes are
activated by engagement of the T cell receptor with
processed antigen fragments presented by professional
antigen presenting cells (e.g. macrophages, dendritic
cells and B cells) [9]. Upon this activation, the tyrosine
kinase Lck becomes activated, and phosphorylates the
intracellular portions of the CD3 complex (called
ITAMs). The most important member of the CD3 family
is CD3-zeta, to which ZAP-70 binds [9].
MUM-1 is a 50 kDa protein encoded by the MUM-1
gene [10]. The MUM-1 molecule also has other
monikers, including 1) interferon regulatory factor 4
(IRF4) and 2) interferon consensus sequence binding
protein for activated T cells (ICSAT). MUM-1 has been
associated with melanocytic cells, and is involved in the
DNA damage response pathway by contributing to the
maintenance of chromatin architecture. Recruited to the
vicinity of DNA breaks by TP53BP1, it plays an
accessory role in facilitating damage-induced chromatin
changes and promoting chromatin relaxation. MUM-1 is
required for efficient DNA repair and cell survival
following DNA damage [10].
We conclude that in this case of a bullous
allergic drug eruption, we observed some degree of
inflammation of the dermal vessels without frank
leukocytoclastic changes. We have described specific
activation markers, especially of the T cell immune
response; and further, overexpression of secondary cell
signaling pathway molecules in dermal blood vessels,
indicating a complex immune response in this patient.
© N Dermatol Online 3.2011
Figure 1.
a-c. H&E shows a subepidermal blister (upper blue arrow) with a positive inflammatory infiltrate of the upper
epidermal vessels (lower blue and maroon arrows). Positive staining of the blister containing FITC conjugated fibrinogen
(white arrow). The blister lumen is shown by yellow stars. d, f. DIF. d. Positive staining of the upper dermal blood vessels
with FITC conjugated anti-human C3(green staining; white arrows). The nuclei of the cells were counterstained with Topro
III (red staining). Blister lumen is indicated by yellow star. f. FITC conjugated anti-human-IgM positive staining of the
upper dermal vessels (green staining, white arrows). anti-human-IgM positive stain of the upper dermal vessels. The nuclei
of the cells were counterstained with Topro III (red staining). Blister lumen is indicated by yellow star. e, g, h, i. IHC. e.
Positive staining with MUM-1 in the small vessels under the blister (brown staining; red arrows). Blister lumen is indicated
by yellow star. g. Positive staining with LAT in the same small vessels as MUM-1, under the blister (brown staining; red
arrows). h. Positive staining with ZAP-70 in the same small vessels as MUM 1 and LAT, under the blister (brown staining;
red arrows). Blister lumen is indicated by yellow star. i. Positive staining with HLA-DPDQDR in upper dermal inflamed
vessels under the blister (brown staining; red arrows). Blister lumen is indicated by yellow star.
106 © N Dermatol Online 3.2011
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Funding source: Georgia Dermatopathology Associates, Atlanta, Georgia, USA
© N Dermatol Online 3.2011